The use of thermostable alpha-amylase as a thinning agent during liquefaction of starch represents a significant improvement in the production of sugar sweeteners. The enzymatic liquefaction of starch substrates reduces the formation of undesirable by-products and also permits the use of a low level of salt which is desirable because a high level of salt in the final glucose syrup involves an additional manufacturing cost for its removal by ion exchange resins. The usefulness of an enzyme such as alpha-amylase for liquefying starch at elevated temperatures depends mainly on the enzyme's thermal stability because exposure to heat causes its irreversible denaturation. This denaturation results in the complete loss of biocatalytic activity. Heat stable alpha-amylases obtained from thermophilic microorganisms have been used to hydrolyze, liquefy and/or convert starch containing materials into starch hydrolyzates. For example, U.S. Pat. Nos. 3,654,081 and 3,912,590 describe the liquefaction of starch at high temperatures using thermostable alpha-amylase produced by bacteria of the species Bacillus licheniformis.
Many of the commercially available alpha-amylase products are obtained from bacterial sources such as Bacillus subtilis, B. licheniformis, B. stearothermophilus, B. coagulans. Fungal alpha-amylases have a very limited application in the liquefaction of starch at high temperatures because they are not generally regarded as thermostable enzymes.
The enzymatic liquefaction of starch is normally carried out at elevated temperatures to increase the susceptibility of the starch to enzymatic hydrolysis. The aforementioned loss of biocatalytic activity can result even with the use of thermostable alpha-amylase at the elevated temperatures preferred for the liquefaction step. Accordingly, stabilizers are added to the enzyme to increase its thermal stability. Thus, it was disclosed by Wallerstein in U.S. Pat. No. 905,029 that the high temperature saccharification of starch by enzymatic means is enhanced by the addition of calcium sulfate to the starch containing medium. The use of calcium ion to thermally stabilize alpha-amylase has become generally accepted in the art but does have certain disadvantages such as the aforementioned problems related to the addition of salts to the starch.
More recently it has been disclosed in U.S. Pat. No. 3,654,081 that starch can be easily and completely liquefied by the addition of calcium and/or sodium salts to an aqueous starch slurry which preferably contains some alpha-amylase.
There is disclosed in U.S. Pat. No. 3,912,590 a process for the combined liquefaction and thinning of starch which involves treating an aqueous starch suspension with alpha-amylase obtained from a bacterium of the species Bacillus licheniformis at an elevated temperature.
U.S. Pat. No. 4,284,722 claims a heat and acid stable alpha-amylase derived from an organism of the species Bacillus stearothermophilus.
Pace et al report in the Journal of Biological Chemistry that the thermal and guanidine hydrochloride denaturation of lysozyme has been investigated at various concentrations of tri-N-acetylglucosamine, which binds specifically at the active site of native lysozyme and that its presence leads to a readily observable stabilization of the protein to thermal and guanidine hydrochloride denaturation.
Chloride ion has been added to aqueous solutions of alpha-amylase which has been found to provide some thermal stabilization although the primary purpose for such addition is pH adjustment.
In U.S. Pat. No. 4,497,897 there is disclosed a method for enhancing the shelf life during storage of protease from Subtilisin Carlsberg which involves the addition of calcium ion and a water soluble carboxylate selected from the group of formate, acetate, propionate and mixtures thereof to a solution of the enzyme.
U.S. Pat. No. 4,318,818 discloses a stabilized aqueous enzyme composition comprising:
(a) from 0 to about 75% of a detergent surfactant; PA1 (b) from about 0.025% to about 10% of pure enzyme, preferably a proteolytic enzyme; PA1 (c) from 0% to about 60% of a low molecular weight primary or secondary alcohol; PA1 (d) from about 0.1% to about 10% of a short chain length carboxylic acid salt, preferably a formate; PA1 (e) a soluble calcium salt in sufficient quantity to provide 0.1 to 10 millimoles of calcium ion per liter; and PA1 (f) the balance being water.
In typical commercial operations, starch is liquefied using thermostable alpha-amylase at a temperature ranging from 80.degree. C. to about 110.degree. C. and at a pH above 6.0. Calcium salts (50-150 ppm Ca.sup.++) are generally added for additional stabilization of the enzyme at the elevated temperatures. These conditions for the enzymatic liquefaction of starch have three disadvantages. First, the hydrolysis of starch above pH 6.0 and at a temperature of 80.degree. C. or above promotes the isomerization of reducing groups of the starch resulting in the formation of maltulose upon subsequent conversion processes which causes a decrease in the final glucose yield. Secondly, the pH optimum of glucoamylase used in the subsequent saccharification of the liquefied starch to produce glucose for subsequent conversion to fructose is generally about 4.5 for Aspergillus type glucoamylases and about 5.0 in the case of Rhizopus type enzymes. Thus, when the starch is liquefied at a pH of 6.0 or above, the pH must be reduced before saccharification using glucoamylase is carried out. This pH adjustment increases the salt content of the resulting glucose syrup due to the addition of acid and consequently increases the expense for ion exchange resins which are required for removal of the excess salt. For the liquefaction to be carried out at a pH of 6.0 or less, additional calcium ion (up to about 500 ppm) must be added. This complicates the complete removal of any added calcium ion from the glucose syrup produced by saccharification of the starch when it is to be used in the production of high fructose corn syrup using glucose isomerase.
It would be desirable, and it is an object of the present invention, to provide a novel method for enhancing the thermal stability of alpha-amylase in aqueous solution. A further object is to provide a stabilized alpha-amylase composition prepared by this method.
An additional object is to provide such a composition which does not require the presence of added calcium ion and can be used to liquefy starch at low levels of calcium (25-50 ppm).
Another object is to provide such a composition which can be used in the high temperature liquefaction of starch at a pH as low as 5.0.